Fig. 2: TLS-associated heterogeneity of T and NK cell states in HNSCC.

a UMAP plot of T/NK cell subclusters identified from scRNA-seq. Subclusters are numbered and colored by identity: CD8⁺ T cells (clusters 1–5), cycling T/NK cells (clusters 6–8), CD4⁺ T cells (clusters 9–14), Treg cells (clusters 15–21), NK cells (clusters 22–26), γδT cells (cluster 27), and ILCs (cluster 28). b Left, GLM-based dot plot showing TLS status-specific enrichment of T/NK cell subclusters. The analysis was conducted using a GLM with a binomial distribution and a logit link function. Estimated marginal means and contrasts were computed with P values indicating the statistical significance of the observed differences. The P values were adjusted using the Bonferroni correction method. A color gradient, transitioning from red (representing enrichment) to blue (representing depletion), encodes the log₂-transformed odds ratios, while the sizes of the depicted points are governed by the Bonferroni-adjusted −log₁₀(P values), highlighting the statistical significance of observed variations. Middle, heatmap of average T cell state scores across T/NK cell subclusters. Right, heatmap of signaling pathway activity scores across T/NK cell subclusters. c Pairwise comparisons of kernel density estimates in UMAP space. The color gradient from red to blue indicates decreasing enrichment of T/NK cells and plasma cells in different TLS statuses. d Images of a mIHC-stained different subclusters of CD8⁺ T cells in HNSCC tumor with different TLS status. Regions with a high density of memory (CCR7⁺CD8⁺), stem-like (TCF1⁺CD8⁺), cytotoxic (Granzyme B⁺CD8⁺), and exhausted (PD-1⁺CD8⁺) CD8⁺ T cells. nTLS and imTLS were repeated four times independently with similar results, mTLS were repeated six times independently with similar results. Scale bars = 100 μm. e PAGA analysis of CD8⁺ T cells. Each color represents a subcluster of CD8⁺ T cell. f UMAP plot colored by pseudotime across subclusters of CD8⁺ T cells. Scaled module scores within these subclusters of CD8⁺ T cells with respect to pseudotime and stem-like, functional, and exhausted markers. g UMAP plot embedding for CD8⁺ T cell subclusters, colored by clone size (upper left) and UMAP embedding for CD8⁺ T cell clusters in different TLS status, colored by cluster, indicating the different clone size by dot size. h Boxplot showed the number of clonotypes of CD8⁺ T cells in different statuses of TLS (n = 4 independent samples nTLS, n = 4 independent samples imTLS, n = 5 independent samples mTLS, one-tailed Mann–Whitney U-test; for box plots: box center line, median; box limits, upper and lower quartiles; box whiskers, maximum and minimum values). i Clone size in each CD8⁺ T cell subclusters separated by TLS status (two-tailed Mann–Whitney U-test). j Heatmap and pie chart showing the number of clonotypes shared between neighboring functional clusters of CD8⁺ T cells in different statuses of TLS. For the heatmap, the color represents the number of shared clonotypes. For the pie chart, the color represents the status of TLS, and the size represents the number of shared clonotypes. k Dot plot showing the overlaps of shared clonotypes between stem-like and functional CD8⁺ T cells, sized by number of clonotypes and colored by TLS status. l Boxplot showing the number of functional clonotypes shared with stem-like CD8⁺ T cells in different status of TLS (n = 4 independent samples nTLS, n = 4 independent samples imTLS, n = 5 independent samples mTLS, one-tailed Mann–Whitney U-test; for box plots: box center line, median; box limits, upper and lower quartiles; box whiskers, maximum and minimum values). m Boxplot showing the proportion of shared stem-like and functional features in expanded clonotypes across different statuses of TLS (n = 4 independent samples nTLS, n = 4 independent samples imTLS, n = 5 independent samples mTLS, one-tailed Mann–Whitney U-test; for box plots: box center line, median; box limits, upper and lower quartiles; box whiskers, maximum and minimum values). Tconv conventional T cell, NK natural killer, mIHC multiplex IHC, ISG interferon-stimulated gene, ILC innate lymphoid cell, Tfh T follicular helper cell, Treg regulatory T cell.